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Mineralogical Magazine, October 2005, Vol. 69(5), pp.

643652

Acidic demineralization of apatites studied by

scanning X-ray microradiography and microtomography

J. C. ELLIOTT*, F. R. G. BOLLET-QUIVOGNE, P. ANDERSON, S. E. P. DOWKER, R. M. WILSON AND G. R. DAVIS


Institute of Dentistry, Queen Mary, University of London, Francis Bancroft Building, Mile End Rd,
London E1 4NS, UK

ABS TR AC T

The mineral in bones and teeth is an impure form of hydroxylapatite (HAP), the principal impurity
being 25 wt.% carbonate. This mineral dissolves during remodelling of bone and also in dental caries
as a result of the action of acids produced by osteoclasts and by bacteria, respectively. In enamel,
demineralization proceeds with preferential loss of carbonate relative to phosphate. Surprisingly, in the
early stages, the demineralization is subsurface. In order to facilitate the understanding of physical
chemical aspects of these processes, we have undertaken studies of demineralization in model systems.
We give three examples here. The first two used scanning microradiography in which the specimen is
stepped across a 1030 mm diameter X-ray beam. Intensity measurements allow calculation of the
mineral mass per unit area in the X-ray path through the specimen. In the first experiment, porous HAP
sections were separated from a reservoir of acidic buffer by a column initially filled with water (the
diffusion length) and scanned with the X-ray beam perpendicular to the axis of the diffusion length.
The rate of total loss of mineral along each profile was calculated from the scans. The rate of
demineralization fell as the diffusion length increased. We believe the explanation is that the rate-
controlling step is the diffusion of dissolved HAP away from the solid to the buffer reservoir. In the
second experiment, demineralizing solution and water were pumped alternately, for equal lengths of
time, past blocks of porous HAP or enamel. The X-ray beam was perpendicular to the exposed surface.
As the rate of switching between solutions decreased, the mean rate of demineralization also fell. We
propose that this effect is due to retention of acid in the pores of the HAP during the time when water
flows, allowing further demineralization to take place during this time. The third study used X-ray
microtomography, a form of 3D microscopy, to study the loss of mineral in compacted carbonate
apatite powders. The powders were packed in six 10 mm internal diameter acrylic cylinders to a depth
of 4 mm (after pressing). One end was covered with a porous polyethylene disc and each tube placed in
acidic buffer for 70 days. Periodic examination by microtomography showed the development of
subsurface demineralization. Infrared spectroscopy of the dissected-out surface layers showed
preferential loss of carbonate over phosphate by comparison with deeper layers. Rietveld analysis of
X-ray powder diffraction data showed changes in the crystallographic structures of the apatites between
the initial and dissected-out apatite.

K EY WORDS :dental enamel, hydroxylapatite, teeth, demineralization, X-ray microtomography, scanning


microradiography.
Introduction
related to hydroxylapatite (Ca10(PO4)6(OH)2,
DENTAL enamel, the most mineralized tissue in the HAP). The vol.% of mineral is usually quoted
body, is made up of ~95 wt.% of an impure as 86 vol.%, but a recent recalculation that used
carbonate-containing apatite (CO3Ap) closely an estimated density of the enamel apatite
(3.0 g cm 3) rather than of HAP (3.16 g cm 3)

yielded 96 vol.% (Elliott, 1997). Enamel apatite is


* E-mail: j.c.elliott@qmul.ac.uk microcrystalline with attened hexagons ~30 nm
DOI: 10.1180/0026461056950276 across and 0.1 to 5 mm or more in length. Most of
# 2005 The Mineralogical Society
J. C. ELLIOTT ET AL.

the CO23 ions replace PO34 ions, but about 10%



is not known if the process that results in these
are thought to replace OH ions in the axis

c changes is the preferential loss of a Mg- and CO3-
channels (Elliott, 1994). A recent Rietveld rich phase in the enamel, or dissolution of a
renement of the structure has been published homogeneous phase, followed by precipitation of
(Wilson et ., 1999). Although there was
al a less soluble apatite with less Mg and CO3.
evidence that CO23 ions replaced PO34 ions, it

However, there is no convincing independent
was not possible to determine the details of the evidence that the apatite in enamel contains a
substitution. However, it2 seems probable that the second, more soluble phase. There is also an
normal to the plane CO3 ion makes an angle of

increased uoride concentration in carious lesions
~30 to the axis, as reported for a synthetic
c (Weatherell ., 1977).
et al
sodium-containing CO3Ap that was studied by In order to try to understand the complex
Rietveld analysis of neutron diffraction powder chemical changes that take place in enamel during
data (Wilson ., 2004). This orientation is also
et al dental caries, many investigators have studied de-
consistent with polarized infrared (IR) measure- and remineralization in model systems that used
ments (Elliott, 1994). either sections or blocks of enamel or HAP
Dental caries is an extremely prevalent disease pellets. We present here three such demineraliza-
that is immensely costly to health services. The tion studies. They have in common the fact that
main chemical change in the formation of enamel they all make use of X-ray attenuation measure-
lesions is the net dissolution of mineral by acids ments. Dowker . (1999) gave an account of
et al
of bacterial origin. An unusual and perplexing related, earlier experiments. Remineralization
feature of the dissolution process is that it takes processes can also be investigated by X-ray
place below the surface of the enamel with the attenuation methods, but generally the effects
retention of a relatively intact surface layer are much more subtle and more difcult to
(Fig. 1). Thus, in enamel lesions, loss of mineral quantify (Gao ., 1993).
et al
may be halted or even reversed through The studies to be presented concern: (1) the
reprecipitation of `mineral' from the saliva. In effect of changes in the diffusion path-length
the early stages of dental caries, there is a between an acidic buffer reservoir and the surface
preferential loss of Mg (Hallsworth ., 1972)
et al of a dissolving apatite pellet. This study
and carbonate (CO3) (Hallsworth ., 1973). It
et al investigated the importance of diffusion processes
in the external solution of a solid dissolving in
acid; (2) the effect of switching periodically
between buffer and water on the mean rate of
dissolution of an HAP porous pellet. A depen-
dence on switching period might be expected if
there was inertia in the system, for example, the
demineralizing solution was retained in the pores
of the solid, so that demineralization continued
after the solution was switched from buffer to
water; and (3) the spatial distribution of the
apatite lost during demineralization of a pressed
pellet of carbonate apatite, including an IR and
powder X-ray diffraction (XRD) study of the
apatite at the end of the demineralization.
FIG. 1. Slice (~15 mm) through a 3-dimensional micro-
tomography image of a human molar tooth. Highly
Experimental

mineralized enamel (light) overlies less well mineralized Apatite and pellet preparation

dentine which surrounds the unmineralized pulp (dark). The HAP was prepared by addition of solutions of
Caries lesions in enamel showing subsurface mineral (NH4)2HPO4 to Ca(NO3)2.4H2O with a Ca/P mole
loss are seen as darker regions adjacent to ssures. To ratio of 1.67 at 8090C and pH 1011
the right, the slice cuts through a ssure; to the left, the (maintained by addition of ammonia solution).
slice cuts through regions of mineral loss that extend in The precipitate was ltered off, washed, dried at
three dimensions from a ssure which terminates in an 80C, then 900C and ground to a ne powder.
adjacent slice. Pellets were prepared by compacting the HAP
644
ACIDIC DEMINERALIZATION OF APATITE

powder in the nger of a surgical glove, 2003; Elliott ., 1994). In this technique, the
et al
isostatically pressing in oil (70 MPa for the X-ray transmission of a narrow X-ray beam
external diffusion study and 275 MPa for the pH through the specimen is measured with a
cycling experiments) followed by sintering in photon-counting detector (Fig. 2) rather than
steam at 900C for 24 h. The porosity for the the conventional photographic emulsion. This
lower-pressure pellets was ~50% and ~20% for has the advantages of avoiding the non-linearity
the high-pressure ones. of emulsions and in particular, allows a much
Seven samples of CO3Ap were prepared by larger dynamic range without problems of
adding 20 g of CaHPO4 to 150 g of ammonium background noise and saturation inherent to
carbonate dissolved in 10 l of deionized water. photographic emulsions. A particular advantage
Stirring at 70C was maintained for 3 days with a of using photon-counting methods rather than an
nal pH of 910. The apatites were separated by emulsion is that the specimen can be some
ltration, washed well, and dried at 100C for distance from the detector, so it can be placed in
24 h. Powder XRD data were collected and a cell with a controlled environment, e.g.
analysed by the Rietveld method as is described temperature. In the present studies, permeable
later. This showed that two of the samples apatite blocks or sections are mounted in cells
contained 1.6 and 0.6 wt.% calcite, but the and immersed in aqueous acidic buffers. The
others contained only well crystallized apatite. sections or blocks are varnished with nail
The sample with 0.6 wt.% calcite was used in the varnish all over, except for a window on one
present study. This CO3Ap contained 11.7 wt.% face or edge so that the acid can diffuse through
CO3 (see Wilson ., 2004 for a description of
et al this exposed face into the solid and cause
the analytical method) and the lattice parameters dissolution. The X-ray beam may be oriented
determined in the Rietveld analysis were: = either perpendicular to the direction of inward
9.4021 A and = 6.8963 A.
a
c diffusion of acid (and outward diffusion of the
products of dissolution), or parallel to the
direction of diffusion (Anderson ., 1998).
et al
Scanning microradiography
With the X-ray beam perpendicular, the pattern
Detailed accounts of scanning microradiography of demineralization through the depth of a
(SMR) have been published (Anderson .,
et al permeable solid can be observed. On the other

FIG. 2. Schematic of scanning microradiographic stage with horizontal and vertical motions. Several cells containing
mineral sections are mounted on the stage so that they can be scanned in any predetermined sequence. Reacting
solutions can be pumped through the cells so that the course of the reaction with the mineral can be studied.
645
J. C. ELLIOTT ET AL.

hand, if the beam is parallel to the direction of 360 (data collection time ~4 h) and recon-
diffusion, changes in the total apatite mineral structed using a cone-beam version of the
along the X-ray path through the depth of the Cartesian axes preprojection algorithm (Davis,
solid are measured, irrespective of the depth at 1998). The nal reconstructed image comprised
which the mineral is lost. Measurements can be 36063506190 cubic voxels of side length
made at a single point, a series of points along a 29.55 mm and represented the 3-dimensional
line or over a 2-dimensional array of points. distribution of the linear attenuation coefcient
Whatever arrangement of points is chosen, at the nominal monochromatic beam energy of
measurements can be repeated sequentially in 40 keV.TMThese images were analysed using the
order to study local changes in the specimen Amira software package (TGS Template
with time. If the changes with time are relatively Graphics Software Inc., USA).
slow, as they usually are, many cells can be For the XMT study, specimens were xed on
mounted on the scanning stage (Fig. 2), the lids of closed plastic bottles to prevent drying
allowing multiple experiments to be undertaken. out during the X-ray exposure. The bottles were
A Hilger & Watts Y33 X-ray generator with a mounted kinematically on the XMT rotation stage
1006100 mm source (after foreshortening) and so they could be removed, the specimen (still
Ag target was used at 40 kV and 1 mA tube attached to the lids) placed in buffer for
current. The aperture comprised two perpendi- demineralization, and then repositioned on the
cular tantalum crossed slits (15 mm gap), posi- stage for re-imaging. Successive scans give a set
tioned 3 cm from the source. X-ray photons were of registered 3-dimensional images of the process
counted with a high purity Ge solid state detector of mineral loss.
coupled with a DSPEC plus (a combined digital
amplier and multichannel analyser), both from
EG&G Ortec (now AMETEK Inc). AgK a IR spectroscopy

photons were selected from the spectrum and Fourier transform infrared (FTIR) spectra were
counted for xed time periods. A PC was used to recorded (Perkin-Elmer Spectrum GX FTIR
control both the counting and movement of the spectrometer) for specimens in KBr discs (2 mg/
SMR stage. 300 mg KBr). Relative areas of the n3 CO3 and n4
PO4 bands were determined with the GRAMS/32
software (Galactic Industries Ltd, Salem, NH,
X-ray microtomography
USA).
A fourth generation X-ray microtomography
(XMT) system (Davis and Elliott, 2003) with a
laboratory X-ray generator (Ultrafocus HMX 160, XRD and Rietveld analysis

X-Tek Systems Ltd, UK, 5 mm source, tungsten Powder XRD patterns were collected following
target, 160 kV max) was used at 90 kV and the procedure described earlier (Wilson .,
256 mA. This system used a cooled slow-scan
et al
1999; Morgan ., 2000). A Ge 111 mono-
CCD camera with a 1:1 lens coupling to a 70 mm chromator giving Cu- a 1 radiation ( l =
et al

1.5406 A) and an INEL CPS-120 curved posi-


K
thick columnar CsI scintillator (Applied
Technologies Ltd, UK). The rotation axis was tion-sensitive detector were used. Samples (typi-
vertical. In order to eliminate ring artefacts, time- cally 5 mg) were spread on a at (711) cut Si
delay integration CCD readout was employed. In single crystal rotating specimen holder, with the
this, the CCD is read out in synchrony with a incoming monochromatic beam striking its
horizontal mechanical motion of the camera surface at an angle of ~3.5, to minimize
across the X-ray beam. Thus the sensitivities of background. Data sets were collected for at least
all the recorded image pixels in the same CCD 1000 min and were calibrated using curves
column were identical as each was an average of produced from lead nitrate (99.999 wt.% purity)
every physical detector element within that usually run before and after each sample. The 29
column. In order to correct for beam hardening, most intense lead nitrate peaks between 19 and
the detector was calibrated with a seven-step 1172y and = 7.8586 A (Nowotny and Heger,
a
aluminium (99.98 wt.%) wedge so that the data 1986) were used for the calibration. Rietveld
appeared to have been collected with a nominally structure renements were made using the
monochromatic beam of 40 keV energy. In the computer program, GSAS (Larson and Von
present study, 600 projections were collected over Dreele, 1986).
646
ACIDIC DEMINERALIZATION OF APATITE

rated by 2 to 4 mm, depending on the cell. Each


line comprised 61 points separated by 40 mm
Effect of external diffusion on the rate of demineralization

Ten 500 mm thick sections were cut from the (10 s counting time per point, typically 20,000
HAP block and varnished except for one edge. photons at points in the solid). Standard
These were mounted in polymethylmethacrylate measurements (30 s counting time, typically
(PMMA) cells (the cross-section of one is shown 130,000 photons) were made outside each
in Fig. 3), with the unvarnished edge uppermost section before and after each line scan for
and thus exposed. The PMMA was used for correction of X-ray generator and detector
making the SMR cells as it is essentially X-ray instabilities. The measurement sequence for the
transparent. The sections were positioned at ten sections took ~6 h and was repeated for
increasing distances from 0 to 9 mm (these 1680 h, except between 600 and 950 h when the
distances will be referred to as diffusion driving mechanism malfunctioned.
lengths, ) from the cell opening. All the cells
l The total mass of HAP lost per unit area of the
were lled with deionized water and then placed section exposed to buffer was calculated for each
in the same large PMMA reservoir as seen in time of measurement and for each section. For
cross-section in Fig. 3. The reservoir was then these calculations, the mass attenuation coef-
lled with1 demineralizing buffer (1 l of cient of HAP for 2Ag- 1 a radiation (22.1 keV) was
K
0.1 mol l acetic acid adjusted to pH 4 with

taken as 4.69 cm g . From this data, the rate of

NaOH). Physical mixing of the solution in the loss of mineral with time was calculated for each
reservoir and in the diffusion length were section, and hence each diffusion length. The
minimized as the reservoir solution was not rates were calculated by linear least-squares
stirred, and thermal gradients were minimized tting a straight line to the data between 300 h
by conducting the study in a temperature- to the end of the experiment (1680 h). As there
controlled room at 252C. were two line scans per section, there were two
Microradiographic scans were made from determinations of the rate of loss of mineral for
outside to within the section in a vertical direction each diffusion length.
(Fig. 3) along two lines. These lines were
perpendicular to the exposed surface and sepa- Demineralization under pH cycling conditions

Five ~1.3 mm thick sections were cut from the


same HAP pellet and varnished except for one
surface. Similarly, ve enamel blocks, again
~1.3 mm thick, were cut from a human molar
tooth, and varnished except for the natural
surfaces. Five SMR cells were made from
PMMA. An enamel section and HAP block
were mounted in each cell so that the X-ray
beam was normal to the unvarnished surface, that
is to say, so that changes in the total apatite
mineral along the X-ray path through the depth of
the solid would be measured. A reservoir was
lled with 50 l of 0.1 mol l 1 acetic acid adjusted

to pH 4 with NaOH. A second reservoir was lled


with 50 l of deionized water. The reservoirs were
connected via four 2-way valves and peristaltic
pumps to each input of four SMR cells. Each cell
had two outputs (emptying and overow) which
FIG. 3. Cross-section of cell with 500 mm thick section were connected via a further four 2-way valves
of HAP in place. The diffusion length, , is the distance and peristaltic pumps to waste. The valves were
computer controlled and programmed so that
l
between the HAP surface and the cell opening into the
buffer reservoir. The rate of demineralization can be buffer and water owed alternately for equal
measured by repeated vertical scanning of the HAP times /2, where is the switching period, with a
T T
section through the X-ray beam. Ten such cells were 30 s emptying after each switching to prevent
mounted in the 1 litre reservoir. contamination of one reservoir solution by the
647
J. C. ELLIOTT ET AL.

other. The values of for the four cells were: 0.5,


T above, this enabled a sequence of XMT images to
1, 2 and 4 h. A constant ow of buffer was be made of the process of demineralization.
pumped through the fth cell to act as a control. The six CO3Ap pellets were demineralized, as
X-ray attenuation measurements through the illustrated in Fig. 4, in acidic buffer1 (100 ml
specimens were made at ve points, immediately sodium acetate/acetic acid, ~0.1 mol l , pH 4.5,

preceded and followed by measurements in the with a thymol crystal) for 70 days with weekly
solution near the sample for normalization replacement of the buffer. The XMT images were
purposes. The counting time was 20 s for all collected at intervals of 1 to 3 weeks. At the end
measurements and the scanning sequence over all of the experiment (70 days), the surface,
specimens (~1 h) was repeated continuously until intermediate, deep intermediate and deep layers
all of the buffer solution had been used up were dissected out from ve of the pellets (the
(~235 h). other pellet was lost during microtomography) for
FTIR spectroscopy and XRD analysis for the
Microtomography study of demineralization in compacted
determination of lattice parameters.
carbonate apatite

Sleeves (10 mm bore61.6 mm wall627 mm Results and discussion

length) and pistons (10 mm diameter 6 45 mm Effect of external diffusion on rate of demineralization

length) were made from PMMA. Six sleeves were Fifteen of the 20 scans showed subsurface
lled with ~0.6 g of CO3Ap each. For loading, a demineralization, a characteristic of natural and
sleeve had a piston put in one end, lled with model caries lesions (Dowker ., 1999). An
et al
CO3Ap, then another piston pressed in from the example is shown in Fig. 5. The other ve scans
other end. One end of the sleeve was then put in showed no subsurface demineralization, although
distilled water so that water was drawn up through there was some evidence of erosive loss of HAP.
the powder without occluding air. Pressing was The most likely explanation for this was the
continued to the limit of the strength of the sleeve. trapping of a small air bubble on the deminer-
The pressed pellet of CO3Ap was then pushed to alizing surface which is known to be an
one end and its exposed surface covered with a occasional problem. This explanation is consistent
disc of porous polyethylene (Fig. 4), which was with the observation that the rates of loss of HAP
retained with a ne nylon thread wound several for these ve scans (marked with crosses in
times over the length of the sleeve. The Fig. 6) were much less than those showing
protruding end of the piston was then cemented subsurface demineralization. As the conditions
to the inside surface of the lid of a plastic bottle. of demineralization for these ve scans were
As described in the microtomography section clearly different from the remaining scans, they
were excluded from the data analysis.
The rates of HAP loss per unit area exposed to
buffer for the different diffusion lengths (in the
range 0.3610 8 to 1.8610 8 g cm 2 s 1)

FIG. 4. Polymethylmethacrylate sleeve (10 mm internal FIG. 5. Scans of a HAP section (only every ~100 h
diameter) with compressed disc of CO3Ap (11.7 wt.% shown) for a diffusion length of 0.2 cm showing
CO3) in buffer at pH 4.5. The exposed surface of the disc subsurface demineralization, a feature typical of natural
has been covered with a porous polyethylene cylinder caries lesions in dental enamel (see Fig. 1). Points are
retained in place with nylon thread (not shown). separated by 40 mm.
648
ACIDIC DEMINERALIZATION OF APATITE

( s i)
R = K C C (1)
where is the rate of dissolution, is the
R K
dissolution rate constant, s the HAP concentra-
C
tion in the demineralizing solution at equilibrium,
and i the concentration of HAP at the solid-
C
interface. At steady state, the rate of dissolution
will equal the rate of diffusion of dissolved HAP
out of the diffusion length ( ). The latter, from
l
Fick's First Law is p i/ , where p is the
D C l D
diffusion coefcient of HAP, and taking into
account: (1) the concentration gradient of HAP in
the diffusion length is constant; and (2) the
concentration of HAP in the reservoir is zero.
Hence
FIG. 6. Rates of total mineral loss for different diffusion
lengths. (*) results with, and (6) results without, Dp
subsurface demineralization. The line is a3 plot of K (Cs Ci ) = Ci 2

equation6 3 2with1 s = 0.00224 g cm


, p = l
4.2610 cm s and = 7.4610 6 cm s 1, the
C D

K

latter two parameters being determined by least-squares Eliminating i between equations 1 and 2 gives
C

tting of equation 3 to the * data points (from Bollet- Dp


Quivogne ., 2005).
et al R=
l + Dp / K
Cs 3

decrease as the diffusion length increases (Fig. 6). C s, the HAP concentration in the deminer-
This suggests that diffusion in the solution alizing solution at equilibrium, was 2.24 g l 1,

external to the dissolving solid controls the rate calculated from the computer program Chemist
of dissolution. (MicroMath Research, Salt Lake City, USA).
A simple model has been used to derive a Using this value, equation 3 was least-squares
quantitative relationship between the steady state tted to the data in Fig. 6, excluding results
rate of dissolution, , and the diffusion length
R marked with crosses. The values for p and D K
(Bollet-Quivogne ., 2005). The assumptions (with standard deviations) were p =
( 4 . 2 0 . 8 ) 661 0 6 c1m 2 s 1 a n d
et al D
made in its derivation were: (1) that the ionization
=
(7.40.6)610 cm s . The value of p is of
K
of the HAP and buffer could be ignored, so they
D
diffused as single species; and (2) that the buffer the expected order of magnitude, though the
concentration at the solid-liquid interface was signicance of the value of is difcult to judge
K
effectively equal to the reservoir concentration because the surface area of the dissolving HAP
soon after the start of, and throughout, the crystals in the face of the HAP aggregate is
experiment. This second assumption was based unknown.
on two propositions: the rst that the buffer
diffuses quickly by comparison with the time-
frame of the experiment primarily because its Demineralization under pH cycling conditions

substantial concentration in the reservoir provides The mass of HAP per unit area integrated along
a signicant gradient for its inward+diffusion; and the X-ray path as a function of time for various
second, that the consumption of H ions required switching periods is given in Fig. 7. The
for the dissolution of HAP at the interface has a intercepts with the vertical axis vary somewhat
negligible effect on the buffer concentration. because of the different initial thicknesses of the
These assumptions are discussed further by samples. The slope of each plot, hence the rate of
Bollet-Quivogne et. (2005) and at http://
al loss of mineral with time, decreases as the
www.smd.qmul.ac.uk/dental/oralgrowdev/ switching period increases, even though the
biophysics/publications/appendix1.doc. mean time of ow of buffer is the same for all
For diffusion-controlled dissolution (Berner, switching periods. The control has a higher rate of
1978) mineral loss because the buffer ows for twice the
649
J. C. ELLIOTT ET AL.

FIG. 7. Mass of HAP per unit area integrated along the X-ray path in hydroxylapatite as a function of time and for
different switching periods ( ). Water and pH 4 buffer owed alternately for equal times with = 0.5, 1, 2 and 4 h.
The plot for continuous demineralization in pH 4 buffer (labelled ?) is also given and crosses over the plot for =
T T
T
at ~140 h.
time. The results for enamel were similar to those transient phenomena that are responsible for this.
for HAP, but the rates of demineralization were The exact nature of these are still to be elucidated,
greater. This is not surprising as, unlike dental but are likely to involve diffusion and volume
enamel, the HAP had been heated to 900C and effects within the subsurface lesion so that buffer
was free of CO3, both of which would reduce the is retained in the pores.
rate of dissolution of HAP in acids.
The results show clearly that the rate of
demineralization increases as the frequency of Microtomography study of demineralization in compacted

switching increases. This implies that deminer- carbonate apatite

alization lasts for a time longer than the actual The volume percent of apatite in the pellets before
time of ow of buffer. There clearly must be some demineralization was ~60%, as determined from

FIG. 8. Left: axial slice (29.5 mm thick) through 3-dimensional microtomography data set of compressed CO3Ap disc
(10 mm diameter) after 46 days in buffer (darker regions correspond to lower concentrations of apatite). Right:
prole of apatite concentration along the white line in the axial slice.
650
ACIDIC DEMINERALIZATION OF APATITE

the mass of apatite used to make the pellet and its


dimensions. Apart from the initial images before
the start of the experiment, all pellets showed
substantial subsurface demineralization and reten-
tion of a well-mineralized surface zone, as
illustrated in Fig. 8 for a pellet after 46 days in
buffer. As demineralization progressed, the
advancing front of the demineralized zone
moved deeper into the pellet, but there was
always some mineral left in the demineralized
zone, as illustrated by the plot in Fig. 8. These
results show that the model system described here
has many of the characteristics of natural caries
lesions, though the details, e.g. the thickness of FIG. 9. FTIR spectrum of apatite from the deep layer
the surface layer, differ. (lower trace) compared with the surface layer (upper
Figure 9 shows the FTIR spectra of the surface trace). Note the lower intensity of the n3 CO3 band at
and deep layers with the major bands identied. ~14001500 cm 1 in the surface layer material.

The n3 CO3 absorption at ~14001500 cm 1 is Samples collected from CO3Ap pellet after 70 days in
buffer at pH 4.5.
clearly of lower intensity in the surface layer
compared with the deep layer, whereas the1
intensity of the n4 PO4 absorption at ~600 cm
deep towards the surface layer. Such changes in
is similar. Plots of the area ratio of the lattice parameters are consistent with there being
n3 CO3/n4 PO4 bands decreased from the original, a structural change associated with the prefer-
outwards to the surface for all the pellets. This ential loss of CO3, rather than dissolution of a
mirrors the preferential loss of carbonate over CO3-rich phase. The actual surface samples
phosphate found in early caries lesions in enamel themselves occupy a completely different position
(Hallsworth ., 1973).
et al in the gure indicating that they are not the end-
Figure 10 shows a plot of the axis the -
a- vs. c member of a progressive alteration. Either there
axis parameters for the unit cells of the original has been some sort of collapse in the unit cell
CO3Ap, deep, intermediate and surface layers. leading to a reduction in the unit-cell parameters,
This clearly shows a linear increase in both the a or the surface layer is the result of reprecipitation
and axes as the sample locus moves from the
c of apatitic material.

FIG. 10. Plot of axis . axis for the original CO3Ap and samples dissected out after 70 days in buffer at pH 4.5.
a vs c
There are only four `Deep' samples because, accidentally, one was not collected.
651
J. C. ELLIOTT ET AL.

Conclusions Research Section A , 410, 329334.


Davis, G.R. and Elliott, J.C. (2003) High denition X-ray
The investigations reported above demonstrate microtomography using a conventional impact X-ray
that X-ray attenuation methods can, or could be, source. Journal de Physique IV , 104, 131134.
used in a wide variety of novel studies of Dowker, S.E.P., Anderson, P., Elliott, J.C. and Gao, X.J.
dissolution processes in apatite-containing (1999) Crystal chemistry and dissolution of calcium
systems (synthetic and biological apatites phosphate in dental enamel. Mineralogical

including bone and dental enamel, whole tissue Magazine , 63, 791800.
or pressed powders). Such studies should give Elliott, J.C. (1994) Structure and Chemistry of the

detailed quantitative information about how the Apatites and Other


Elsevier, Amsterdam.
Calcium Orthophosphates.

dissolution of apatites is affected by chemical Elliott, J.C. (1997) Structure, crystal chemistry and
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CIBA Foundation Symposium, 205. John Wiley &
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Studies of remineralization are also possible, but Elliott, J.C., Anderson, P., Gao, X.J., Wong, F.S.L.,
much more difcult. Although this work was Davis, G.R. and Dowker, S.E.P. (1994) Application
restricted to apatites, the experimental approach of scanning microradiography and X-ray micro-
might be extended to a wide range of materials of tomography to studies of bones and teeth. Journal of
geological and mineralogical interest, and need X-ray Science and Technology , 4, 102117.
not even be restricted to acidic dissolution Gao, X.J., Elliott, J.C., Anderson, P. and Davis, G.R.
processes. (1993) Scanning microradiographic and microtomo-
graphic studies of remineralisation of subsurface
enamel lesions. Journal of the Chemical Society,
Acknowledgements
Faraday Transactions , 89, 29072912.
This research was supported by the Medical Hallsworth, A.S., Robinson, C. and Weatherell, J.A.
Research Council programme grant G9824467 (1972) Mineral and magnesium distribution within
and subsequently by the St Bartholomew's and the approximal carious lesion of dental enamel.
the Royal London Charitable Foundation grant Caries Research , 6, 156168.
RAB 03/PJ/18. The microtomography was Hallsworth, A.S., Weatherell, J.A. and Robinson, C.
supported by the Engineering and Physical (1973) Loss of carbonate during the rst stages of
Sciences Research Council grant GR/R28911/01. enamel caries. Caries Research , 7, 345348.
Larson, A.C. and Von Dreele, R.B. (1986) GSAS,
General Structure Analysis System . Los Alamos
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652